1. Field of the Invention
The present invention concerns a method of preventing the transmission of herpesvirus or treating patients infected by herpesvirus and chlamydia trachomatis by administration of lysine-containing proteins or peptides modified by a lysine modifying agent, such as an aromatic acid anhydride compound, or arginine-containing proteins or peptides modified by an arginine modifying agent.
2. Background Information
Herpesviruses include the following viruses isolated from humans:
(1) herpes simplex virus 1 ("HSV-1") PA0 (2) herpes simplex virus 2 ("HSV-2") PA0 (3) human cytomegalovirus ("HCMV") PA0 (4) varicella-zoster virus ("VZV") PA0 (5) Epstein-Barr virus ("EBV") PA0 (6) human herpesvirus 6 ("HHV6") PA0 (7) herpes simplex virus 7 ("HSV-7") PA0 (8) herpes simplex virus 8 ("HSV-8")
Herpesviruses have also been isolated from horses, cattle, pigs (pseudorabies virus ("PSV") and porcine cytomegalovirus), chickens (infectious larygotracheitis), chimpanzees, birds (Marck's disease herpesvirus 1 and 2), turkeys and fish (see "Herpesviridae: A Brief Introduction", Virology, Second Edition, edited by B. N. Fields, Chapter 64, 1787 (1990)).
Herpes simplex viral ("HSV") infection is generally a recurrent viral infection characterized by the appearance on the skin or mucous membranes of single or multiple clusters of small vesicles, filled with clear fluid, on slightly raised inflammatory bases.
The herpes simplex virus is a relatively large-sized virus. HSV-2 commonly causes herpes labialis. HSV-2 is usually, though not always, recoverable from genital lesions. Ordinarily, HSV-2 is transmitted venereally.
The time of initial herpes simplex virus infection is usually obscure except in the uncommon primary systemic infection occurring in infants and is characterized by generalized cutaneous and mucous membrane lesions accompanied by severe constitutional symptoms. Localized infections ordinarily appear in childhood, but may be delayed until adult life. It is presumed that the herpes simplex virus remains dormant in the skin and that herpetic eruptions are precipitated by overexposure to sunlight, febrile illnesses, or physical or emotional stress; also, certain foods and drugs have been implicated. In many instances, the trigger mechanism remains undetected.
The lesions caused by herpes simplex virus may appear anywhere on the skin or on mucous membranes, but are most frequent on the face, especially around the mouth or on the lips, conjunctiva and cornea, or the genitals. The appearance of small tense vesicles on an erythematous base follows a short prodromal period of tingling discomfort or itching. Single clusters may vary from 0.5 to 1.5 cm in size, but several groups may coalesce. Herpes simplex on skin tensely attached to underlying structures (for example, the nose, ears or fingers) may be painful. The vesicles may persist for a few days, then begin to dry, forming a thin yellowish crust. Healing usually occurs within 10 days after onset. In moist body areas, healing may be slower, with secondary inflammation. Healing of individual herpetic lesions is usually complete, but recurrent lesions at the same site may result in atrophy and scarring.
In females infected with HSV-2, there may be no skin lesions, the infection may remain entirely within the vagina. The cervix is frequently involved, and there is increasing evidence that this may be a factor in the development of carcinoma of the cervix.
Corneal lesions commonly consist of a recurrent herpetic keratitis, manifest by an irregular dendritic ulcer on the superficial layers. Scarring and subsequent impairment of vision may follow.
Gingivostomatitis and vulvovaginitis may occur as a result of herpes infection in infants or young children. Symptoms include irritability, anorexia, fever, inflammation, and whitish plaques and ulcers of the mouth. Particularly in infants, though some times in older children, primary infections may cause extensive organ involvement and fatal viremia.
In women who have an attack of HSV-2 late in pregnancy, the infection may be transmitted to the fetus, with the development of severe viremia. Herpes simplex virus may also produce fatal encephalitis.
Kaposi's varicelliform eruption (eczema herpeticum) is a potentially fatal complication of infantile or adult atopic eczema. Exposure of patients with extensive atopic dermatitis to persons with active herpes simplex should be avoided.
With respect of prophylaxis of HSV, in patients in whom sunlight is a precipitating factor, avoidance of overexposure and application of a sunscreen preparation is advisable.
No local or systemic chemotherapeutic agent has been demonstrated to be effective for treating herpes simplex virus with the possible exception of topical idoxuridine (IDU) in superficial herpetic keratitis. Reports on this compound in cutaneous herpes are conflicting. Other drugs which have been employed to treat HSV include trifluorothymidine, vidarabine (adenine arabinoside, ara-A), acyclovir, and other inhibitors of viral DNA synthesis may be effective in herpetic keratitis. These drugs inhibit herpes simplex virus replication and may suppress clinical manifestations. However, the herpes simplex virus remains latent in the sensory ganglia, and the rate of relapse is similar in drug-treated and untreated individuals. Moreover, some drug-resistant herpes virus strains have emerged.
The utilization of condoms may provide a degree of protection against transmission of HSV-2 infection during sexual intercourse, but a difficulty arises when condoms are not employed. Moreover, the use of condoms appears to be a culturally and socially unacceptable practice in many countries.
Although men may be able to protect themselves from HSV-2 infection by using condoms, women who are sexually active have no similar means. Women can encourage their male sex partners to use a condom, but may not succeed. The female condom, which is just becoming available, is expensive and there is presently no evidence that it prevents transmission of HSV-2.
Even maintaining a monogamous sexual relationship is no guarantee of safety, for if a woman's male partner becomes infected, he can pass the virus to her, and as more women are infected, so are more babies.
Diseases caused by varicella-zoster virus (human herpesvirus 3) include varicella (chickenpox) and zoster (shingles).
Cytomegalovirus (human herpesvirus 5) is responsible for cytomegalic inclusion disease in infants. There is presently no specific treatment for treating patients infected with cytomegalovirus.
Epstein-Barr virus (human herpesvirus 4) is the causative agent of infectious mononucleosis and has been associated with Burkitt's lymphoma and nasopharyngeal carcinoma.
Animal herpesviruses which may pose a problem for humans include B virus (herpesvirus of Old World Monkeys) and Marmoset herpesvirus (herpesvirus of New World Monkeys).
In an effort to expand the diversity of compounds with medically useful biological activities, the chemical transformation of synthetic peptide-based or other combinatorial libraries of organic compounds have been recently conceived (Ostresh, J. M., Husar, G. M., Blondelle, S. E., Dorner, B., Weber, P. A., and Houghten, R. A., (1994), "Libraries from Libraries: Chemical Transformation of Combinatorial Libraries to Extend the Range and Repertoire of Chemical Diversity," Proc. Natl. Acad. Sci. USA, 91, 11138-11142). Such transformations can be accomplished with reagents which alter chemical moieties of library constituents in a defined manner and high yield. The diversity of compounds of interest to medicinal chemistry can be also increased by applying the concept of chemical modification to natural products, either in the form of mixtures of compounds or in the form of isolated individual components.
Site-specific chemical modification of amino acid residues in proteins has been widely used in structure/function studies in which a loss or decrease of biological activity was related to chemical modification of specific amino acid residues. Methods for covalent chemical modification of C, M, H, K, R, W, Y residues and carboxyl groups were described and applied to many proteins (Lundblad, R. C., (1991), Chemical Reagents For Protein Modification, CRC Press, Boca Raton, Fla.). In a few cases, it was reported that changes in net electric charge caused by chemical modification of proteins increased their activity "cationized" protein antigens were reported to have increased or altered immunogenicity (Muckerheide et al., 1987; Suzuki et al., 1992)! or mimicked changes occurring during in vivo protein turnover, causing binding of the chemically modified proteins to scavenger receptors on cells, (Westwood, M. E., McLellan, A. C., and Thornalley, P. J., (1994), "Receptor-mediated Endocytic Uptake of Methylglyoxal-modified Serum Albumin. Competition with Advanced Glycation End Product-modified Serum Albumin at the Advanced Glycation End Product Receptor", J. Biol. Chem., 269, 32293-32298; Lo, T. W. C., Westwood, M. E., McLellan, A. C., Selwood, T., and Thornalley, P. J., (1994), "Binding and Modification of Proteins by Methylglyoxal Under Physiological Conditions. A kinetic and Mechanistic Study with N.alpha.-Acetylarginine, N.alpha.-Acetylcysteine, and N.alpha.-Acetyllysine, and Bovine Serum Albumin", J. Biol. Chem., 269, 32299-32305; Abraham, R., Singh, N., Mukhopadhyay, A., Basu, S. K., Bal, V., and Rath, S., (1994), "Modulation of Immunogenicity and Antigenicity of Proteins by Maleylation to Target Scavenger Receptors on Macrophages," J. Immunol., 154, 1-8).
Heretofore U.S. Pat. No. 5,164,486 and U.S. Pat. No. 5,256,412 (hereinafter collectively referred to as "Tsunoo et al") disclosed an anti-HIV agent comprising a plasma protein of which the polarity of at least one amino group was chemically modified into a negatively charged moiety by using aliphatic acid anhydrides. Tsunoo et al discussed treatment, not prevention of HIV infection, preferably by intravenous administration. Tsunoo et al mentioned maleic anhydride and succinic anhydride, but did not discuss aromatic acid anhydrides. The treated proteins blocked fusion of infected cells with uninfected cells by blocking HIV-1 mediated fusion. Tsunoo et al described plasma proteins such as human serum albumin, human immunoglobulin, human transferrin and human fibrinogen, but did not discuss milk, casein or whey.
Jansen et al (WO 92/15316) and "Potent In Vitro Anti-Human Immunodeficiency Virus-1 Activity of Modified Human Serum Albumins", Molecular Pharmacology, 39, 818-823 (1991)) described the use of cis-aconitic anhydride, propane-1,2,3-tricarboxylic acid anhydride, acetic anhydride, propionic anhydride, butyric anhydride, glutaric anhydride, phthalic anhydride, and maleic anhydride to modify protein and polypeptides from proteins such as albumin to prepare anti-viral pharmaceuticals, by imparting a negative charge to the proteins or polypeptides. The examples in WO 92/15316 were carried out only with aliphatic acid anhydrides.
However, to the best of applicants' knowledge, there have not been reported attempts of others to systematically modify protein amino acid residues of one or more kinds in order to: (1) generate compounds with medically important biological properties, of which the original protein was totally devoid; and (2) produce at the same time a compound(s) having the particular biological activity optimized.
Chlamydiae are a large group of obligate intra-cellular parasites closely related to gram-negative bacteria. They are divided into 2 species, Chlamydia psittaci and Chlamydia trachomatis, on the basis of antigenic composition, intracellular inclusions, sulfonamide susceptibility, and disease production.
C trachomatis produces compact intracytoplasmic inclusions that contain glycogen; it is usually inhibited by sulfonamides. It includes agents of mouse pneumonitis and several human disorders such as trachoma, inclusion conjunctivitis, nongonococcal urethritis, salpingitis, cervicitis, pneumonitis of infants, and lymphogranuloma venereum.
In endemic areas, sulfonamides, erythromycins, and tetracyclines have been used to suppress chlamydiae that cause eye infections. Periodic topical application of these drugs to the conjunctivas of all members of the community is sometimes supplemented with oral doses; the dosage and frequency of administration vary with the geographic area and the severity of endemic Trachoma. Drug-resistant C trachomatis has not been definitely identified except in laboratory experiments. Even a single monthly dose of 300 mg of doxycycline can result in significant clinical improvement, reducing the danger of blindness. Topical application of corticosteroids is not indicated and may reactivate latent trachoma. Chlamydiae can persist during and after drug treatment, and recurrence of activity is common.
It is believed that over 400 million people throughout the world are infected with trachoma and that 20 million are blinded by it. The disease is most prevalent in Africa, Asia, and the Mediterranean Basin, where hygienic conditions are poor and water is scarce. In such hyperendemic areas, childhood infection may be universal, and severe, blinding disease (resulting from frequent bacterial superinfections) is common. In the USA, trachoma occurs sporadically in some areas, and endemic foci persist on Indian reservations.
Control of trachoma depends mainly upon improvement of hygienic standards and drug treatment.
When socioeconomic levels rise in an area, trachoma becomes milder and eventually may disappear. Experimental trachoma vaccines have not given encouraging results. Surgical correction of lid defermities may be necessary in advanced cases.
C trachomatis, immunotypes D-K, is a common cause of sexually transmitted diseases that may also produce infection of the eye (inclusion conjunctivitis). In sexually active adults, particularly in the USA and western Europe--and especially in higher socioeconomic groups--C trachomatis is a prominent cause of nongonococcal urethritis and, rarely, epididymitis in males. In females, C trachomatis causes urethritis, cervicitis, salpingitis, and pelvic inflammatory disease. Any of these anatomic sites of infection may give rise to symptoms and signs, or the infection may remain asymptomatic but communicable to sex partners. Up to 50% of nongonococcal or postgonococcal urethritis or the urethral syndrome is attributed to chlamydiae and produces dysuria, non-purulent discharge, and frequency of urination.
This enormous reservoir of infectious chlamydiae in adults can be manifested by symptomatic genital tract illness in adults or by an ocular infection that closely resembles trachoma. In adults, this inclusion conjunctivitis results from self-inoculation of genital secretions and was formerly thought to be "swimming pool conjunctivitis."
A neonate can acquire the infection during passage through an infected berth canal. Inclusion conjunctivitis of the newborn begins as a mucopurulent conjunctivitis 7-12 days after delivery. It tends to subside with erythromycin or tetracycline treatment, or spontaneously after weeks or months. Occasionally, inclusion conjunctivitis persists as a chronic chlamydial infection with a clinical picture indistinguishable from subacute or chronic childhood trachoma in nonendemic areas and usually not associated with bacterial conjunctivitis.
It is essential that chlamydial infections be treated simultaneously in both sex partners and in offspring to prevent reinfection.
Tetracyclines (eg, doxycycline, 100 mg/d by mouth for 10-20 days) are commonly used in non-gonococcal urethritis and in nonpregnant infected females. Erythromycin, 250 mg 4-6 times daily for 2 weeks, is given to pregnant women. Topical tetracycline or erythromycin is used for inclusion conjunctivitis, sometimes in combination with a systemic drug.
Genital chlamydial infection and inclusion conjunctivitis are sexually transmitted diseases that are spread by indiscriminate contact with multiple sex partners. Neonatal inclusion conjunctivitis originates in the mother's infected genital tract. Prevention of neonatal eye disease depends upon diagnosis and treatment of the pregnant woman and her sex partner. As in all sexually transmitted diseases, the presence of multiple etiologic agents (gonococci, treponemes, Trichomonas, herpes, mycoplasmas, etc) must be considered. Instillation of 1% silver nitrate into the newborn's eyes does not prevent development of chlamydial conjunctivitis. The ultimate control of this--and all sexually transmitted disease depends on reduction in promiscuity, use of condoms, and early diagnosis and treatment of the infected reservoir.
Adults with inclusion conjunctivitis often manifest upper respiratory tract symptoms (e.g., otalgia, otitis, nasal obstruction, pharyngitis), presumably resulting from drainage of infectious chlamydiae through the nasolacrimal duct. Pneumonitis is rare in
Neonates infected by the mother may develop respiratory tract involvement 2-12 weeks after birth, culminating in pneumonia. There is striking tachypnea, paroxysmal cough, absence of fever, and eosinophilia. Consolidation of lungs and hyperinflation can be seen by x-ray. Diagnosis can be established by isolation of C trachomatis from respiratory secretions and can be suspected if pneumonitis develops in a neonate who has inclusion conjunctivitis. Systemic erythromycin (40 mg/kg/d) is effective treatment in severe cases.
Lymphogranuloma venereum ("LGV") is a sexually transmitted disease, characterized by suppurative inguinal adenitis, that is common in tropical and temperate zones. The agent is C trachomatis of immunotypes L1--L3.
Untreated LGV infections tend to be chronic, with persistence of the agent for many years. Little is known about active immunity. The coexistence of latent infection, antibodies, and cell-mediated reactions is typical of many chlamydial infections.
The sulfonamides and tetracyclines have been used with good results to treat LGV, especially in the early stages. In some drug-treated persons there is a marked decline in complement-fixing antibodies, which may indicate that the infective agent has been eliminated from the body. Late stages require surgery.
LGV is most often spread by sexual contact, but not exclusively so. The portal of entry may sometimes be the eye (conjunctivitis with an oculoglandular syndrome). The genital tracts and rectums of chronically infected (but at times asymptomatic) persons serve as reservoirs of infection.
Although the highest incidence of LGV has been reported from subtropical and tropical areas, the infection occurs all over the world.
The measures used for the control of other sexually transmitted diseases apply also to the control of LGV. Case-finding and early treatment and control of infected persons are essential.